This application supports the movement of basic research on new drug regimens against human colon cancer to the clinic. Specifically, we will focus on the selective control of pyrimidine metabolism to reduce the toxicity and/or to enhance the efficacy of 5-Fluorouracil (5-FU). Our preliminary results have demonstrated that Brequinar potentiated the cytotoxicity of 5FU in mice bearing colon tumor 38 by selectively increasing the effect of 5-FU on the tumor. We will investigate the biochemical mechanisms underlying the apparent efficacy of this regimen and extend the study of this combination to other in vivo models: a murine colon tumor cell line (CT-26) which is resistant to fluoropyrimidines, and a human adenocarcinoma cell line (CX-l). We have also compared the effects of orotic acid on uridine pools in both normal and malignant cells in mice bearing colon tumor 38. These studies demonstrated that orotic acid selectively increased UXP pools in normal tissues compared to tumor. The use of orotic acid allowed increased doses of 5-FU to be given in tumor bearing animals and resulted in an improved therapeutic effect. We propose to study the incorporation of orotic acid into the Brequinar plus 5-FU regimen with the goal of further increasing the therapeutic index. We will also study the biochemical mechanisms accounting for these observations and determine the antitumor effect of this new combination in additional in vivo models. In parallel with the animal studies, we will carry out studies in patients using specimens obtained by cryosurgery to determine the biochemical effect of low doses of Brequinar on uridine and UXP pools in colorectal tumors and adjacent normal tissue in patients with metastatic colorectal cancer. We will determine the plasma pharmacokinetics of uridine and Brequinar and the concentrations of Brequinar in both tumor and adjacent normal tissue. These data will be used to design future clinical trials that combine Brequinar and 5FU with or without orotic acid.